Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of Y. pestis parent and mutant strains. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. We have also established in vitro methods to infect fleas and to monitor transmission dynamics of fleas over a one-month period following their infectious blood meal. With this system we are able to compare the relative importance of the two modes of transmission and the relative vector competence of different flea species. Our studies of flea vector competence and vectorial capacity will be useful to develop more realistic mathematical modeling of the epidemiology of plague transmission and the conditions that lead to plague epizootics. During FY2018, we reported that efficiency with which fleas transmit Y. pestis can vary significantly depending on the source of their infectious bloodmeal. Fleas infected using rat blood transmit better than fleas infected using mouse blood, especially during the first week after infection (early-phase transmission). The enhanced transmission efficiency correlated with the propensity of rat hemoglobin to crystallize in the flea gut and lead to heavier colonization of the foregut. These results are consistent with our proposed regurgitative mechanism for early-phase transmission. The relative insolubility of the hemoglobin of rats and the Sciurid family of rodents (e.g. ground squirrels, prairie dogs, and marmots) and the slower digestion of their blood appears to promote regurgitative transmission, which may be one reason why these rodents are particularly prominent in plague ecology. We implemented new experimental systems to maintain and monitor infection status and transmission efficiency of individual fleas at different times after infection. The data are being used to estimate values for important parameters such as the probability of flea vectors developing a transmissible infection after feeding on a bacteremic host and the transmission efficiency during a four-week period after infection. Limited data are available are currently for these values, which are needed for understanding plague epidemiology. In collaboration with Dr. Angela Luis at the University of Montana, we have developed mathematical models to better comprehend the key conditions that give rise to periodic plague epizootics, and are using our experimentally derived data in these models to compare the relative importance of biofilm-independent (early phase) and biofilm-dependent transmission mechanisms. We have developed standardized methods to more rigorously quantify vector competence parameters (broadly including infectivity, flea foregut blockage rate, transmission rate, and transmission efficiency) throughout a 4-week period following a single infectious blood meal); and with the help of colleagues at the USGS and elsewhere have been evaluating four flea species considered to be poor vectors. For example, the prairie dog flea Oropsylla hirsuta had been claimed to rarely become blocked or transmit beyond the early phase, and recently published models argued that early-phase transmission was the driving force behind plague epizootics in those rodents. Prairie dog colonies are subject to periodic explosive plague epizootics that can essentially extirpate the colony, which pose a public danger to rural communities and hinder efforts to reintroduce the black-footed ferret, an endangered species. The conditions that give rise to prairie dog epizootics are enigmatic. Based on one limited 1940 study, the predominant flea of prairie dogs, O. hirsuta, is a poor vector. Because of this, alternate transmission routes in addition to the classic flea-borne route have been proposed, none of which add up. Similarly, Dr. James Belthoff, Boise State University, is providing Pulex irritans fleas, which we also evaluated. This flea is also considered to be a poor vector but has controversially been hypothesized to have transmitted Y. pestis from human to human during the European plague epidemics of the Middle Ages. We confirmed that the human flea P. irritans is a poor vectors, but that O. hirsuta is a more efficient vector than previously recognized. In all flea species examined, high transmission efficiency correlated with the development of biofilm-dependent proventricular blockage. The early-phase transmission efficiency was low in all species. Reliable vector competence data regarding these fleas will enable more realistic modeling of these epizootiologic/epidemiologic scenarios. Working with the RML Genomics Unit, we have completed in vivo (flea digestive tract) transcriptional profiling of Y. pestis, Y. pseudotuberculosis wild-type (unable to form biofilm in the flea proventriculus), and a Y. pseudotuberculosis mutant strain that is able to produce proventricular-blocking biofilm in fleas. Transcriptomic profiling has been performed on RNA of bacteria recovered from the digestive tract of fleas 1 day and 14 days after the infectious blood meal. Two methods were used: 1) an improved microarray that includes all conserved and unique Y. pestis and Y. pseudotuberculosis ORFs, pseudogenes, and intergenic regions (both strands); and 2) small RNA deep sequencing (RNA-Seq). Surprisingly, the Y. pseudotuberculosis mutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change expression levels of the hmsHFRS genes responsible for the biofilm matrix. The Y. pseudotuberculosis mutant uniquely expressed much higher levels of one of the Yersinia Type 6 secretion systems (T6SS-4) in the flea, and this locus was required for flea blockage by Y. pseudotuberculosis, but not by Y. pestis. Major differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing related genes, and stress response genes were evident during flea infection. The results provide insights into how Y. pestis has adapted to life in its flea vector and point to evolutionary changes in the regulation of biofilm development pathways.